N-hydroxylating monooxygenases (NMO) are members the class B flavoprotein monooxygenases. They catalyze the N-hydroxylation of lysine and ornithine and play and essential role in the biosynthesis of hydroxamate containing siderophores. Siderophores are high affinity iron-chelators composed of catechol and hydroxamate functional groups that are synthesized and secreted by several microorganisms and plants. It has been showed that many NMOs are essential for virulence in many opportunistic pathogens such as Aspergillus fumigatus and Pseudomonas aeruginosa. The focus of my research is on the N-hydroxylating enzymes: Siderophore A (SidA) from Aspergillus fumigatus and Amycolatoposis alba monooxygenase (AMO). One of my projects is focusing on identifying inhibitors of SidA that will ultimately block the siderophore biosynthesis in A. fumigatus. Out of 973 compounds screened using an activity high-throughput assays two compounds were identified. These were, wortmannin a steroid metabolite and ebselen a benzoselenazole as SidA inhibitors with IC50 values of 369 µM and 11 µM respectively. A second part of this works investigates the hydroxamate formation of the siderophore albachelin in Amycolatoposis alba with the purpose of better understanding this class of enzymes and their catalytic mechanism. The enzyme was purified and characterized in its holo (FAD-bound) and apo (unbound) forms. Pre-steady and steady state kinetics shows that the two forms have different coenzyme preference; apo-AMO prefers NADH while holo-AMO has a higher affinity to NADPH. / Master of Science in Life Sciences / N-Hydroxylating monooxygenases (NMOs) are unique class of enzymes which are involved in the production of small iron binding molecules known as siderophore. Siderophores are used by some fungi and bacteria to acquire iron from the human host during infection. This thesis focuses on the inhibition and characterization of Siderophore A (SidA) from Aspergillus <i>fumigatus</i> and <i>Amycolatopsis alba</i> monooxygenases (AMO). SidA is an enzyme of interest because it is required for siderophore production and has been shown to be required for pathogenesis in <i>A. fumigatus</i>. In this thesis, a drug-discovery processes known as highthroughput screening was used to identify potential inhibitors of SidA. Using a several other assays, two compounds were validated as inhibitors of <i>A. fumigatus</i>. In addition, this work also investigates the reaction of AMO by focusing on the different stages of the enzyme mechanism; such as oxygen consumption, product formation, and flavin reduction. Overall, the goal of this thesis is to provide insight into the N-hydroxylating monooxygenases class of enzymes as well as to find new compounds that can lead to the next generation of antifungal drugs.